Plastids and mitochondria are double membrane-bound organelles found in eukaryotic cells. Chloroplasts, plastids containing the green pigment chlorophyll, are the most complex of the plant membranous organelles. Both chloroplasts and mitochondria specialize in the synthesis of ATP, using energy derived from electron transport from photosynthetic phosphorylation in chloroplasts and from oxidative phosphorylation in mitochondria.
To perform their role in the cell, plastids must continuously import all types of molecules, including proteins. The biogenesis and development of plastids require the coordinated assembly of plastidic- and nuclear-encoded proteins which are incorporated into membranes or other parts of the plastid. The process by which nuclear-encoded plastid proteins are targeted from the site of synthesis to the site of function is mediated by a complex series of events involving a multitude of proteinaceous signals and factors located in the cytosol and the plastidic compartment. Few of these factors are known and the molecular infrastructure underlying this important and complex event is far from being understood.
Chloroplast envelope proteins play a major role in modulating the vectorial flow of molecules across the membrane, including large proteinaceous entities. The import of proteins into the plastid is a complex process requiring the close collaboration of both the outer envelope and the inner envelope membranes. Evidence for the possible existence of two distinct protein import complexes, one in each envelope membrane, is beginning to emerge from a number of recent investigations (Waegemann, K. and Soll, J. (1991) Plant J. 1:149-158; Soll, J. and Waegemann, K. (1992) Plant J. 2:253-256; Schnell, D. and Blobel, G. (1992) J. Cell. Biol. 120:103-115; Alefson, H., Waegemann, K. and Soll, J. (1994) J. Plant Physiol. 144:339-345; Schnell, D., et al. (1994) Science 266:1007-1012; Kessler, F., et al. (1994) Science 266:1035-1039; Wu, C., Seibert, F. S. and Ko, K. (1994) J. Biol. Chem. 269:32264-32271).
An important step in the characterization of the protein translocating complexes is the identification of the components involved. The identification of outer and inner plastid envelope polypeptides has been accomplished using a variety of strategies (Ma, Y., et al. (1996) i J. Cell Biol. 134:315-327; Cornwall, K. L. and Keegstra, K. (1987) Plant Physiol. 85:780-785; Kaderbhai, M. A., et al. (1988) FEBS Lett. 232:313-316; Pain, D., et al. (1988) Nature 331:232-237; Schnell, D., et al. (1990) J. Cell Biol. 111:1825-1838; Hinz, G. and Flugge, U.-I. (1988) Eur. J. Biochem. 175:649-659; Soll, J. and Waegemann, K. (1992) Plant J. 2:253-256; Waegemann, K., et al. (1990) FEBS Lett. 261:89-92; Perry, S. E. and Keegstra, K. (1994) Plant Cell 6:93-105; Alefson, H., et al. (1994) J. Plant Physiol. 144:339-345; Schnell, D. J., et al. (1994) Science 266:1007-1012; Kessler, F., et al. (1994) Science 266:1035-1039; Wu, C., et al. (1994) J. Biol. Chem. 269:32264-32271; Hirsch, S., et al. (1994) Science 266:1989-1992; Seedorf, M., et al. (1995) Plant J. 7:401-411; Seedorf, M. and Soll, J. (1995) FEBS Lett. 367:19-22; Gray, J. C. and Row, P. E. (1995) Trends Cell Biol. 5:243-247). To date, these studies collectively indicate that envelope proteins with molecular masses of 21, 30, 34, 36, 44, 45, 51, 66, 70, 75 86, 97 and 100 kDa may be possible constituents of the plastid protein import apparatus; however, it is not obvious from the existing data whether some of the predicted similar sized components are identical to each other. Further, it is not known if any of the components have an active role in protein transport.
A mechanism for controlling the transport of substances into plastids could be used for modification of plastid pathways and products which occur in particular tissue types, such as the starch and fatty acid biosynthesis pathways in roots and seeds. Major drawbacks to plastid modification of this caliber, however, are the limited knowledge of genes encoding plastid transport proteins and the lack of characterization of such proteins.
Further, plastid transport mechanisms could be usefully incorporated into other organisms, especially prokaryotes. The heterologous production of protein pharmaceuticals in Escherichia coli is a cornerstone of the biotechnology industry. The technology provides an attractive and viable means for the production of proteins in quantities and qualities that are otherwise expensive and difficult to obtain from natural sources.
The gene sequence and encoded protein of one plastid membrane component has been identified. Ko, K., et al. (1995) J. Biol. Chem. 270:28601-28608; GenBank.TM./EMBL Data Bank, accession no. X79091. However, no role in transport was determined for this protein.
To date, no one has reported eukaryotic transport gene function or the functioning of a transport gene from a eukaryotic organelle in prokaryotic cells. An additional transport gene in both prokaryotic and eukaryotic cells would be useful to increase translocation and expression of cellular products. Increased incorporation of proteins into membranes to elevate membrane function would also be desirable.